Lower Aliphatic Alcohols Application of the Zerewitinoff Determination WILLIAM HOLLYDAY AND D. L. CO’M’LE, Rutgers University, New Brunswick, N. J.
T
H E problem of the application of the Zerewitinoff determination to the lower aliphatic alcohols aroused the authors’ interest when they attempted to use a Kohler Grignard machine (6, 7’) for a Zerewitinoff determination on a low boiling alcohol. Kohler’s directions did not involve the use of a solvent for the unknown but because of the much greater volatility of their unknown, as compared with the examples reported by Kohler, they elected to use isoamyl ether as a solvent for the unknown as well as for the Grignard reagent. Discordant results followed and a literature study revealed that Hibbert (4) had obtained “rather startling results” with methanol, ethanol, and 1-propanol, dissolved in isoamyl ether and in phenetol, although his values for a-naphthol were satisfactory. Odd0 (10) also reported that isoamyl ether, anisole, toluene, and ligroin sometimes gave low values, but he and other workers (2, 12) obtained good results by dissolving the alcohols in pyridine and in ethyl ether (1). With the exception of Ciusa, all the workers mentioned worked with the Grignard reagent in isoamyl ether.
Relation between Concentration and Gas Evolution The use of isoamyl ether might have been suspected as the source of the troubles of Hibbert and the authors, except that Kohler seldom had any solvent but isoamyl ether present in his application of the method to substances of higher molecular weight. A systematic investigation involving isoamyl ether soon demonstrated that the concentration of the alcohol in the reaction mixture was the disturbing factor. With methanol a t 0.245 mole per liter of reaction mixture, 89 per cent of the theoretical amount of gas was obtained, a t 0.100 molar, 94 per cent, and a t 0.0647 molar, 99.9 per cent. The amount of gas produced appeared to depend inversely on the amount of methoxymagnesium iodide that precipitated, because a t the higher concentration a large amount of precipitate, sticky and gelatinous in appearance, was present, whereas a t 0.0647 molar the amount was very slight. When methanol, ethanol, and 1-propanol are treated in concentrations of about 0.23 molar the results are lowest for methanol, intermediate for ethanol, and theoretical for 1-propanol; the precipitates are, respectively, heavy, intermediate, and light. Kohler (7) observed that the formation of insoluble products not infrequently makes it impossible to complete the reaction. Hibbert (8) recently suspected a precipitate of interfering with the determination of active hydrogen and carbonyl addition in vanillin, which was dissolved in isoamyl ether, and Haurowitz (3) suggested that benzoic acid gave only 66 per cent of the ethane expected on treatment with diethyl zinc because the benzoic acid formed a complex with the resulting zinc benzoate. The data submitted by Zerewitinoff (11, 12) and Flaschentrager indicate that concentrations of their alcohols were all below 0.1 molar and with the additional advantage of the greater solubility of polar compounds in pyridine their satisfactory results are readily understood. The present work indicates that the critical concentration in isoamyl ether reaction mixtures for methanol is about 0.07 molar, for ethanol is about 0.1 molar, and for 1-propanol may be above 0.24 molar. I n the present work a concentration effect was also noticed on the amount of gas given by acetophenone. Whereas Kohler (7)recorded 15 per cent of the theoretical for one active
hydrogen a t a probable molarity of 0.56, the authors obtained 13 per cent a t 0.221 molar, 15 per cent a t 0.133,22 per cent at 0.0753, and 23 per cent a t 0.0653. I n similar machines others (8) have obtained values of 12 per cent for no solvent, 3 per cent for xylene, and 78 per cent for dioxane and pyridine without noting the concentrations.
Loss of Alcohol on Sweeping Apparatus with Nitrogen At corresponding concentrations Hibbert’s values were much lower than the authors’, for methanol 46 per cent against their 89 per cent, for ethanol 73 per cent against 101 per cent, and for 1-propanol 83 per cent against 100 per cent. Obviously the formation of a precipitate cannot account for all of Hibbert’s error, especially in the case of 1-propanol where, by the authors’ experience, the formation of a precipitate was slight. An explanation was found in a detail of Hibbert’s experimental procedure (4, 6) in which the sample in solution was placed in the flask and “the air in the flask was displaced by dry nitrogen” which was led into the flask by a tube that reached nearly to the surface of the solution. This procedure undoubtedly swept some of the alcohol vapors out of the apparatus, inasmuch as in a similar procedure the authors found that 5 ml. of isoamyl ether lost 21.4 mg.; after addition of 39.3 mg. of methanol, and sweeping a t the same rate for the same length of time, the loss was 50.7 mg. If it is assumed that the loss of isoamyl ether was the same in both instances, approximately 75 per cent of the methanol was lost during the sweeping. Hibbert’s losses could not have been as great, but these results indicate a probable source of error. The lower values were to be expected from the more volatile alcohols, a prediction that is supported by Hibbert’s data.
Effect of Heating on Yield of Gas A study was also made of the effect of heating and time of reaction on the amount of gas produced. Ten minutes’ im-
TABLEI. EFFECTOF HEATINQ Molarity of Methanol in Reaction Mixture 2 1 1 3 5 5 5 5 4 6 9 10 8 11 11 11 13 15
12 16 14 18 19 17
0.245 0.226 0.226 0.167 0.162
0.162
0.162 0.162 0.142 0.114 0.104 0.103 0.102 0.100 0.100 0.100 0.0647 0.0618 0.0591 0.0578 0.0575 0.0532 0.0528 0.0519
70 of Theory of Methane
Time.of Standing before Heating Min.
89.0 86.4 95.6 92.8 81.7 89.0 89.7 93.2 93.1 96.3 94.2 95.4 97.0 94.1 99.8 103.0 99.9 100.8 101.9 97.9 100.8 102.2 99.4 98.4
30 30 30 30 10 360 480 480 630 13 55 5 600 3 3 3 10 10 10 11 7 8 10 13
Time of Heating
Total Time
Min.
Min.
10
None 10
18 None None None 10
None 17 15 17
None 10 10 10
10 17 10 15 6 10 10 25
60 30 60 60 10 360 480 510 630 60 120 60 600 60 1260 4080 60 60 60 60 60 60 60 120
ANALYTICAL EDITION
October 15, 1942
h
Alr N2
--
0
775
and G enclose approximately 105 ml. of gas, depending on which particular flask, E, is attached. A simple calculation indicates that the temperature of the ordinary laboratory is not likely to change sufficiently during the usual analysis to affect the results seriously. The particular advantage of an unjacketed apparatus is that it may be mounted on a single large ring stand and thus made extremely portable and less fragile. The obvious disadvantages of changes in temperature and in pressure in operating over long periods of time were overcome by measuring the volume of gas enclosed in the reaction chamber, E,the lead tubes, i and m,the drying tube, G, and down to the zero mark on the gas buret. This volume was designated as X and determined with the aid of the mercury auxiliary tube, M , as follows: With a flask attached to the machine and with H connected to n the mercury level was lowered until near the 20-ml. mark of the buret. The machine was left to come to room temperature for a few minutes and stopcock Z was turned to connect first va and n and then m and H. Again the machine was allowed to stand several minutes and the mercury level observed, to make certain the whole was a t room temperature. The barometric pressure was observed. A pressure greater or less than atmospheric was then exerted on the enclosed gases by allowing mercury to flow in or out of the auxiliary leveling tube, M . A cathetometer was used to determine the difference in heights of the levels of mercury in the two columns. The volume, X, may then be calculated using the equation:
where PI is the initial and atmospheric pressure, P1 is the final pressure imposed by the leveling bulb, z is the final buret reading, and y is the buret reading made at the beginning of the determination. The per cent of the theoretical volume of gas evolved was determined with the aid of the following equation:
FIGURE 1 . APPARATUS
mersion of the reaction flask in a beaker at 70" to 100" C. was sufficient when the concentration of the alcohol was correct. Long periods of standing, u p to 10.5 hours in the case of methanol, did not produce a theoretical yield of gas when the concentration of the alcohol was incorrect (Table I). Preliminary experiments, in which the temperature was raised to 130" by means of an oil bath, also did not serve in lieu of a proper concentration.
Absorption of Oxygen by Reagent If air is present within the apparatus, oxygen will be absorbed by the Grignard reagent and low results will be obtained. In all the methods in the literature, this difficulty has been eliminated (a) by replacement of the air with some indifferent gas such as nitrogen ( 5 , 10,11), methane, or ethyl ether vapor ( I ) , or (b) by allowing the oxygen of the air within the apparatus t o be absorbed before proceeding with the reaction liberating the hydrocarbon (2, 9, 11). Experience during this work indicated that different preparations of methylmagnesium iodide absorbed oxygen at different rates and that a simplified procedure wherein air is used directly is not practical. Tank nitrogen, without further purification of any kind, provided a satisfactory atmosphere. However, the authors have had insufficient experience with tank nitrogen to justify extending its use to cover the original purpose of the "machine", in which Kohler used tank nitrogen purified by passage through a train consisting of ten parts in order to preserve a standardized Grignard solution for months.
Experimental MODIFICATION ASDUSE OF MACHINE.The apparatus used is similar to Kohler's, with the exception that (see Figure 1) Z is now a 3-way 120" cock, c is a simple 2-way rock, the drying tube contains calcium chloride, an auxiliary mercury column is attached to the gas buret, and none of the parts are jacketed. E, i, m,
where M is the molecular weight of the alcohol, Tz is the temperature a t the beginning of the experiment, W is the weight of the alcohol in grams, and R is 62,400 (in mm. of mercury times ml. per mole per degree). The meaning of the other symbols is the same as above. The procedure adopted after the discovery that tank nitrogen could be used as an inert atmosphere was to weigh the alcohol in about 40 ml. of isoamyl ether and dilute it to 50 ml., using a volumetric flask. After sweeping the apparatus with tank nitrogen, including the empty reaction chamber, E an aliquot part of the alcohol solution was added from buret D, !olloned by the methylmagnesium iodide, allowance being made for the 0.1-ml. portion of alcohol solution which always remained in the bore of stopcock C. After addition of the reagent, the flask was heated by a 600ml. beaker of hot water, heated to boiling in another part of the room. After 10 minutes' heating, E was cooled to room temperature with a beaker of cool water, the beaker removed, E wiped dry, and the apparatus allowed to stand until the mercury column in the gas buret indicated that thermal equilibrium had been reached-generally 5 to 10 minutes. Reference is made to the original articles on the machine (1) for additional details. OTHERALCOHOLANALYSES.I n seven determinations with ethanol, commercial absolute reagent, a t molarities running from 1.7 per cent; four deter0.222 to 0.201 the average was 98.5 minations a t molarities from 0.0810 to 0.0754 gave 102.1 =!= 1.4 per cent. I-Propanol from Eastman, fractionally redistilled (b. p. 96.897.2' a t 767 mm., ny 1.386), gave in experiments 2 to 11 a t molarities from 0.238 to 0.225 an average of 100.3 =t1.4 per cent. Exprriment 1 for an unknown reason gave only 92.8 per cent. Allyl alcohol from Eastman was fractionally distilled (b. p. 96.5-96.7' a t 760.6 mm., ny 1.413) and in five determinations gave an average of 100.0 6 0.6 per cent. tert-Butyl alcohol from Eastman was fractionally recrystallized, m. p. 25.3-25.7", and gave in experiments 2 to 4 an average of 101.4 * 0.9 per cent with experiment 1 giving 105.3 per cent. The methanol used in obtaining the data in Table I was fractionally redistillrd Baker and Adamson grade, absolute (b. p. 64.8-64.9" a t 762.4 mm., n y 1.329).
Summary The lower aliphatic alcohols react quantitatively with methylmagnesium iodide, isoamyl ether being used as t h e
776
INDUSTRIAL AND ENGINEERING CHEMISTRY
solvent for both the substance being investigated and the Grignard reagent. From a practical standpoint the complete reaction must take place in a reasonable length of time, which requires that the reaction mixture be so dilute that a precipitate does not interfere by keeping the reactants from each other. Heating at a high temperature or very long standing is not a satisfactory suhstitute for a proper concentration of the alcohol in the reaction mixture. The apparatus described by Kohler and co-workers has been modified as to stopcocks and jacketing and made much more portable, The use of tank nitrogen without further purification is suggested as an inert atmosphere for the Zerewitinoff determination.
Vol. 14, No. 10
Results are given for analyses using methanol, ethanol, 1propanol, tert-butyl alcohol, allyl alcohol, and acetophenone.
Literature Cited ~chim ~ itaz., ~ so, . 11, 53 (1g20).
(1) ciusa, R.,G ( 9 ) Flaschentragcr, 2. phyrioz. Chem.. 146, 219 (1925). (3) Haurowitz, F., Mikrochemie, 7, 88-93 (1929).
~~~~~~~~,"ddc~~7s;;d,0,0;~~~~
(1904), (6) Kohler a n d Richtmeyer. J . Am. Chem. SOC.,52, 3737 (1930). (7) Kohler, Stone. and Fuson, Ibid., 49, 3181 (1927). (8) Lieff, Wright, and Hibbert. Ibid., 61, 865 (1939). (') Marian and Marian, Biochem. J * * 746 (193")* (10) Oddo, Ber., 44, 2048 (1911). ( 1 1 ) Zerewitinoff,Ibid,, 40, 2023 (1907). (12) Ibid., 45, 2384 (1912). 241
Determination of Soybean Flour in Sausages and Other Meat Products A Protein Separation Method JOHN BAILEY, Illinois Department of Agriculture, Division of Foods and Dairies, Chicago, Ill.
S
AUSAGE and similar meat food products are prepared from meats and meat by-products that are usually pickled and then cooked and smoked. They may contain a little spice, cereals, starch, milk powder, and eggs (16, 68), and also salt, nitrates, nitrites, sugars, and sodium benzoate (67). The regulations provide that the combined amount of cereal, starch, flour, and milk powder in sausages shall not exceed 3.5 per cent (66), and in certain meat products it is also necessary to know, for label requirements, whether more or less than 5 per cent has been added (66). Soybean flour is now finding a use in these products, and food control officials and members of the industry realize that it will eventually be necessary t o develop a method of analysis for determining the percentage in these products.
Available Methods To analyze such a complex mixture for the amount of soybean flour, some particular constituent of the soybean must be found which will differentiate the soybean from all the other ingredients. This constituent or a definite part of it must withstand the manufacturing process of the sausage, as the cooking and smoking, and also its own manufacturing process in being converted into flour, as the oil expression or extraction. La Wall and Harrisson presented a test based on thp enzyme urease, which may be detected by the liberation of ammonia from urea (36). This is limited to sausage samples where the urease has not been destroyed or inactivated. and later the authors advised ronfirming the test by identifying the characteristic cell structures (87). On the other hand, it has been recommended that, the enzymes of soybean flour be inactivated by heating to a sufficiently high temperature for better processing (18). Kerr recommended these tests as qualitative, pointed out some limitations, and concluded that a quantitative method is urgent (3s). In locating the hourglass or I-shaped rells with the microscope, it is best to examine with polarizcd liAht, under which they take on a certain sheen. The present-day methods of preparing flour make it almost impossible to locate a single cell even with polarized light. Assuming that thcre is little or no nitrogen-free extract in meat, excepting liver products, but a considerable amount in soybean, Hayward reported on a calculating method (14), and also on an immunological method developed by Glynn which is
based on a quantitative precipitin test (II). On the former test no further report was made by the A. 0. A. C., while on the latter a detailed study was recently published (7). The method appears to be specific for soybean but it requires time for the production of a satisfactory serum and the zone of optimal precipitation may shift because of the presenre of other ingredients. A method based on the determination of insoluble nonfermentable sugars was proposed by Hendrey (15). After the sausage is givrn a preliminary treatment to remove moisture and fat, the soluble sugars are removed with 50 per cent alcohol, and the remaining carbohydrates are hydrolyzrd with hydrorhlnric acid and t h e n fermented nith yeast. The sugars remaining after the fermentation are determined and multiplied by a factor. This method was tried out by Lythgoe d a / . (38).who concluded that it is sufficiently accurate for law-enforccment purposes but not specific for eoybcan. For some time the present author has been working on the chemical separation of the soybean protein and has rendered several reports, but has withheld publication because the method was not previously adaptable to liver sausage or t o meat products that contained liver.
Theory Soybean or soy flour is prepared by finely grinding soybeans, removing the hulls, and debittering, usually by a steaming process. The flour may contain all the fat of the soybean but is generally low in fat, seldom containing above 6 or 7 per cent. Soybean flour may be considered an offspring of the soybean oil meal. Commercial flours in 1915 gave a n average protein content of 42.5 per cent (6S), while oil meals prepared by present-day manufacturing methods give slightly higher figures (71). The chief protein of soybean is a globulin, glycinin, so named by Osborne and Campbell (63). A great part of this protein dissolves in a water solution of the ground seed,. probably owing t o the presence of phosphates, and is precipitated by the addition of a little acid in the form of white spheroids. Some other properties of glycinin are given in Table I. The principle of the method is based on the separation of glycinin or a definite part of it from all the other proteins found in meats and in other binders and vegetable substances included in meat products, Glycinin can be boiled in a water solution with little coagulation and the remaining sol-
